The breast cancer susceptibility gene BRCA2 is expressed in a wide range of tissues as an 11-kb mRNA transcript that encodes a 3418-amino acid protein involved in the response to DNA damage. To obtain better a molecular characterization of BRCA2 expression in sporadic breast cancer, we quantified BRCA2 mRNA by means of RT – PCR in a large series of human primary breast tumours. BRCA2 expression showed wide variations in tumour tissues, being underexpressed in 14/127 (11%) and overexpressed in 25/127 (20%). BRCA2 overexpression (but not underexpression) correlated significantly with Scarff, Bloom and Richardson (SBR) histopathological grade III (P=0.007) and was mainly attributed to nuclear polymorphism (P=0.005) and mitotic index (P=0.048), suggesting that the BRCA2 gene contributes to the proliferation rate in breast tumours. BRCA2 status (under and/or overexpression versus normal expression) was not associated with subsequent relapse and with significantly shorter disease-free survival. The observed disruption of BRCA2 expression is not due to allelic loss, because the latter did not correlate with altered BRCA2 mRNA expression in our tumour series. Taken together, these data suggest the involvement, especially by overexpression, of the BRCA2 gene in sporadic breast tumours, and the existence of another important tumour-suppressor gene in breast cancer, in the 13q12-q13 region.
Breast cancer, one of the most common life-threatening diseases in women, occurs in hereditary and sporadic forms. The two major breast cancer susceptibility genes, BRCA1 and BRCA2, have recently been isolated (Miki et al., 1994; Wooster et al., 1995). Both behave as classical tumour-suppressor genes, loss of both alleles being required for malignancy. Recent studies provide a role for BRCA1 and BRCA2 proteins in response to DNA damage, by interaction with RAD51, a protein involved in the repair of double-strand DNA breaks, as well as in mitotic and meiotic recombination (Zhang et al., 1998). A number of germline mutations in the BRCA1 and BRCA2 genes have been identified in families prone to breast cancer (Shattuck-Eidens et al., 1995; Couch et al., 1996; Phelan et al., 1996).
BRCA2, like BRCA1, is not frequently mutated in sporadic breast cancers (Lancaster et al., 1996; Miki et al., 1996; Teng et al., 1996). However, high frequencies of loss of heterozygosity (LOH) on 17q12-q21 and 13q12-q13 (sites of BRCA1 and BRCA2) (Cleton-Jansen et al., 1995; Kerangueven et al., 1995; Beckmann et al., 1996; Hamann et al., 1996; Kelsell et al., 1996; Bièche et al., 1997) point to a significant role or these two genes in the genesis of sporadic breast cancer, through a mechanism other than structural mutation. LOH at BRCA2 was found to be an independent prognostic factor (Van Den Berg et al., 1996). Such sporadic involvement is also convincing in regard to the BRCA1 results of Thompson et al. (1995) which showed down-regulation of BRCA1 mRNA levels in tumours relative to normal breast epithelial cells.
To clarify the significance of the BRCA2 gene in the development of sporadic breast cancer, we quantified BRCA2 gene expression by means of competitive RT – PCR in a series of 127 unilateral invasive primary breast tumours. We also determined whether alterations of BRCA2 expression correlate with LOH at the BRCA2 locus.
BRCA2 mRNA expression in normal breast tissues
To determine the cut-off point for altered BRCA2 expression in breast cancer tissue, the NBRCA2 value, calculated as described in Materials and methods, was determined for 16 normal breast RNA samples. As this value consistently fell between 0.6 and 1.6 (mean 0.94±0.21), values of 2 or more were considered to represent overexpression, and values of 0.5 or less underexpression, of BRCA2 mRNA.
BRCA2 mRNA expression in tumour breast tissues
Among the 127 breast tumour RNA samples tested, 39 (31%) showed altered BRCA2 mRNA expression. Fourteen tumours (11%) showed underexpression of BRCA2 mRNA (NBRCA2 from 0.1 – 0.4) and 25 (20%) showed overexpression (NBRCA2 from 2.2 – 13). Figure 1 represent data on tumours in which the BRCA2 gene was overexpressed (BRCA07, NBRCA2=4.44), expressed normally (BRCA76, NBRCA2=1.06) and underexpressed (BRCA43, NBRCA2=0.16). The BRCA2 primer pair used in this study was placed in the 3′-terminal region (exons 24 – 26) of the BRCA2 gene; similar results were obtained with a second BRCA2 primer pair in the 5′-region (exons 3 – 5) in 30 of the 127 breast tumours analysed in this study (data not shown).
Correlation between BRCA2 mRNA levels and clinical and pathological parameters
We sought links between BRCA2 mRNA level status and standard clinicopathological and biological factors in breast cancer (Table 1). The only statistically significant association was between BRCA2 mRNA overexpression and histopathological grade III (P=0.007). Sixty per cent (15/25) of tumours with overexpression of BRCA2 were histopathological grade III, compared to 30.8% (4/13) of those with underexpression and 30.9% (25/81) of those with normal expression of BRCA2.
Histoprognostic grade is a combination of three criteria: tubular differentiation, nuclear polymorphism, and mitotic index. We therefore also tested these three components in relation to the BRCA2 mRNA level (Table 2). The prevalence of histopathological grade III in the BRCA2 mRNA overexpressed tumours was attributable to nuclear polymorphism (P=0.005) and mitotic index (P=0.048), but not to tubular differentiation.
Moreover, patients with tumours overexpressing and/or underexpressing BRCA2 did not relapse more frequently (Table 1) and did not have significantly shorter disease-free survival after surgery (log-rank test), compared to patients with tumours normally expressing BRCA2.
Relationship between BRCA2 mRNA levels and 13q12-q13 LOH
The 127 tumours studied for BRCA2 expression had also been tested for LOH at four polymorphic DNA marker loci flanking BRCA2 (D13S289, D13S260, D13S171, D13S267). All patients were informative for one or more loci on 13q12-q13. LOH was found in 44.1% (56/127) of the tumour DNAs, while the remainder had a normal profile. As shown in Table 3, we found no link between 13q12-q13 LOH and BRCA2 mRNA overexpression, underexpression or both.
Germ-line mutations of the two breast cancer susceptibility genes BRCA1 and BRCA2 appear to be responsible for approximately 50 and 20% of hereditary breast cancers, respectively. The supposition that BRCA2 is a tumour-suppressor gene is supported by the observation that most breast tumours arising in patients with germ-line BRCA2 mutations exhibit LOH at the BRCA2 locus, typically involving loss of the wild-type BRCA2 allele (Collins et al., 1995; Gudmundsson et al., 1995). However, similar to the failure to find BRCA1 mutations in sporadic breast cancers, only a small percentage of sporadic breast tumours appear to carry somatic mutations in the BRCA2 gene despite the fact that 30 – 40% of sporadic breast cancers show LOH at this locus (Cleton-Jansen et al., 1995; Kerangueven et al., 1995; Beckmann et al., 1996; Hamann et al., 1996; Kelsell et al., 1996; Bièche et al., 1997). It is possible that DNA lesions not detectable by standard mutation screening may be responsible for disrupting the function of BRCA2 in sporadic tumours. Little is known about BRCA2 gene expression, except that BRCA2 mRNA has a tissue-specific expression pattern similar to that of BRCA1 mRNA and that these two genes are coordinately regulated during mammary epithelial proliferation and differentiation, despite the fact that they display no homology (Rajan et al., 1997). To find out more about BRCA2 mRNA expression in sporadic breast cancer, we quantified BRCA2 gene expression by means of competitive RT – PCR in a series of 127 unilateral invasive primary breast tumours. Surprisingly, we observed both underexpression (11%) and overexpression (20%) of BRCA2 mRNA. Interestingly, a significant link was observed between BRCA2 mRNA overexpression and tumours of histopathological grade III (P=0.007), suggesting that overexpression of BRCA2 plays a role in the aggressiveness of breast tumours. The prevalence of histopathological grade III in the BRCA2 mRNA overexpressed tumours was mainly attributed to nuclear polymorphism and mitotic index, which respectively reflect extent of genetic changes in tumours and the proliferation rate. These results obtained in vivo are thus in agreement with reports from several authors (Rajan et al., 1996; Vaughn et al., 1996) showing that BRCA2 mRNA expression is up-regulated in rapidly proliferating cells in vitro. These observations are consistent with a model in which BRCA2 exerts a positive effect on proliferation. Such a model is difficult to reconcile with the genetic evidence that BRCA2 is a tumour-suppressor gene whose overexpression should inhibit proliferation and vice versa. Rajan et al. (1997) suggested the possible existence of a regulatory loop in which proliferation induces BRCA2 expression that, in turn, inhibits proliferation. The existence of such a feedback loop would imply that the up-regulation of BRCA2 expression induced by proliferation is a protective response tending to decrease breast cancer progression. Interestingly, among the 44 patients with grade III tumours, only 20% (3/15) of those with BRCA2 overexpression relapsed, as compared with 41.4% (12/29) of patients with normal or subnormal BRCA2 expression, suggesting that BRCA2 mRNA level status could have prognostic significance in this specific subset of grade III breast tumours.
We observed no link between BRCA2 expression status and steroid-receptor status, although it has previously been shown that mRNA BRCA1 and BRCA2 expression is up-regulated by estrogen in human breast cancer cell lines (Spillman and Bowcock, 1996). However, it has also been shown that the upregulation of BRCA1 mRNA expression by ovarian hormones in the mouse mammary gland is significantly greater than that of BRCA2 (Rajan et al., 1997).
We also clearly identified 14 tumours with underexpressed BRCA2 mRNA. This subset of breast tumours, where no prevalence of histological grade III was observed, is distinct from the group with BRCA2 overexpression. It has been shown that BRCA2 may play a part in monitoring genomic integrity and in DNA repair. Low expression of BRCA2 mRNA could thus be the cause of the genetic instability observed in human breast tumours. This hypothesis should be tested in a larger group of tumours with BRCA2 underexpression, by studying genetic instability (as measured in terms of gene amplifications and LOHs).
Disruption of expression of BRCA2 mRNA observed in this series of sporadic breast tumours was not due to the loss of one BRCA2 allele, we failed to observe any link between LOH at BRCA2 and altered BRCA2 mRNA expression, suggesting that LOH did not affect the level of BRCA2 mRNA. This result raises two points. (a) BRCA2, in sporadic breast tumours, does not seem to act as a classical tumour-suppressor gene that is inactivated when both alleles acquire alterations (somatic mutations and/or allele loss, typically through deletion). It is important to note that the promoter of BRCA2 showed absence of methylation of CpG dinucleotides in breast tumours (Collins et al., 1997), suggesting that this alternative mechanism of tumour-suppressor-gene inactivation by transcriptional regulation of mRNA expression is unlikely to explain the differences in BRCA2 mRNA expression observed in this study; (b) the BRCA2 gene itself is not the main target gene of the high level (44% in this study) of LOH at 13q12-q13 in breast cancer. This raises the possibility that another key tumour-suppressor gene in this region plays an important role in breast cancer.
In conclusion, this study suggests that the BRCA2 gene is involved in sporadic breast cancer, especially in cell proliferation. Our findings should be confirmed at the protein level when suitable antibodies become available. Further studies are also necessary to elucidate the genetic (or epigenetic) mechanism responsible for the observed dysregulation of BRCA2 mRNA expression.
Materials and methods
Patients and samples
We analysed tissue from excised primary breast tumours from 127 women treated at the Centre René Huguenin from 1977 to 1989. The samples were examined histologically for the presence of tumour cells. A tumour sample was considered suitable for this study if the proportion of tumour cells was more than 60%. Immediately following surgery the tumour samples were stored in liquid nitrogen until extraction of RNA and high-molecular-weight DNA.
The patients (mean age 58 years, range 34 – 91) met the following criteria: primary unilateral non-metastatic breast carcinoma on which complete clinical, histological and biological data were available; and no radiotherapy or chemotherapy before surgery. The main classifying prognostic factors are presented in Table 4. The median follow-up was 8.9 years (range 0.2 – 16.2). Forty-five patients relapsed (distrubution of first relapse events among patients was as follows: 13 local and/or regional recurrences, 28 metastases, and four both).
Specimens of adjacent normal breast tissue were taken from seven breast cancer patients, and normal breast tissue was obtained from eight women undergoing cosmetic breast surgery, as a source of normal RNA. Total RNA from a pool of six normal human breast tissue samples was also purchased from Clontech.
Peripheral blood leukocytes were used as a source of normal DNA for each patient.
Evaluation of ‘classical’ prognostic factors
The histological type and steroid-hormone receptor status of each tumour, and the number of positive axillary nodes, were established at the time of surgery. The malignancy of infiltrating carcinomas was scored according to Bloom and Richardson's (1957) histoprognostic system. Estrogen and progesterone receptor status was assayed as described by the European Organisation for Research and Treatment for Cancer (EORTC, 1980), with a detection threshold of 10 fmol/mg cytosolic protein.
A quantitative RT – PCR method was used to examine BRCA2 mRNA expression. The precise amount of total RNA added to each reaction (based on optical density) and its quality (i.e. lack of extensive degradation) are both difficult to assess. We therefore also quantified the transcripts of TBP (a component of the transcription factor TFIID) as the endogenous mRNA control, and each sample was normalized on the basis of its TBP content. We selected the TBP gene as an endogenous control because the prevalence of its transcripts is similar to that of the BRCA2 target gene, and because there are no known TBP retropseudogenes. (Retropseudogenes lead to co-amplification of contaminating genomic DNA and thus interfere with RT – PCR, despite the use of primers in separate exons). We therefore rejected the β-actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HPRT (hypoxanthine phosphoribosyltransferase) genes as endogenous controls because of the existence of corresponding retropseudogenes (Dirnhofer et al., 1995; Sellner and Turbett, 1996); we also rejected the human 18S rRNA gene, which is intronless and has very high abundance of transcripts; and the β2-microglobulin gene whose expression seems altered in some tumours (Mullen, 1998).
Total RNA was extracted from breast specimens by the acid-phenol guanidium method (Chomczynski and Sacchi, 1987). The quality of the RNA samples was determined by electrophoresis through denaturing agarose gels and staining with ethidium bromide, and the 18S and 28S RNA bands were visualized under ultraviolet light. The yield was quantified spectrophotometrically in a 50 μl microcuvette.
RNA was reverse transcribed in a final volume of 20 μl containing 1× RT – PCR buffer (1 mM each dNTP, 5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3), 20 units of RNase inhibitor, 50 units of Moloney Murine Leukaemia Virus reverse transcriptase (Perkin-Elmer), 2.5 μM random hexamers and 1 μg of total RNA. The samples were incubated at 20°C for 10 min and 42°C for 30 min, and reverse transcriptase was inactivated by heating at 99°C for 5 min and cooling at 5°C for 5 min. The volume was then made up to 200 μl with 1× RT – PCR buffer, yielding the RT mix used for PCR. Ten microliters (1/20 of RT-mix) was used for subsequent PCR.
Primer pairs for quantitative RT – PCR were chosen with the assistance of the Oligo 4.0 (National Biosciences) computer program and by performing BLASTN searches against dbEST and nr (the non redundant set of GenBank, EMBL and DDBJ database sequences) to confirm the total gene specificity of the nucleotide sequences chosen for the primers and the absence of polymorphisms.
BRCA2-specific primers used for PCR amplification were the following: BRCA2-9469U (5′-GTTGTGAAAAAAACAGGACTTG-3′) and BRCA2-9821L (5′-CAGTCTTTAGTTGGGGTGGA-3′); the BRCA2-specific product size corresponds to 353 bp. Primers specific for amplification of a 260-bp product of the TBP gene were TBP-750U (5′-ACAGGAGCCAAGAGTGAAGAA-3′) and TBP-1009L (5′-CCAGAAACAAAAATAAGGAGA-3′).
Primers are designated by the nucleotide position (relative to BRCA2 GenBank #U43746 and TBP GenBank # X54993) corresponding to the 5′ position, followed by the letter U for upper (sense strand) or L for lower (antisense strand). To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons or in a different exon. For example, the upper primer of BRCA2 (9469U) was placed at the junction between exons 24 and 25, whereas the lower primer (9821L) was placed in exon 26. One member of each primer pair was labeled with fluorescein at the 5′ end by using the fluoro-prime method.
BRCA2 mRNA steady-state level
To take into account PCR variability, quantitative RT – PCR is based on co-amplification of specific cDNA with a ‘quantitative DNA standard’ (QDS), using the same two primers (Foley et al., 1993; Pannetier et al., 1993). Specific QDS were generated by PCR using the ‘looped oligo’ method (Sarkar and Bolander, 1994), with creation of a 12-bp insert in the PCR product sequence for each test gene (BRCA2 or TBP), as described by Lazar et al. (1995). With this procedure, target cDNA and QDS yield labelled PCR products of different sizes, which we identified by using a DNA sequencer. Gene mRNA values were expressed as a ratio between the target cDNA and its corresponding QDS. Amplification efficiency between target cDNA and QDS during exponential and non-exponential phases was checked. According to Pannetier et al. (1993), the ratios between the target cDNA and its QDS are similar whatever the number of cycles.
For each PCR run, a master mix was prepared on ice with 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 400 nM each primer, a known quantity of the corresponding QDS and one unit of AmpliTaq DNA polymerase (Perkin Elmer). The amount of QDS was selected for each target cDNA so as to obtain similar fluorescence intensities for standard and corresponding cDNA-derived PCR products. Ten microliters of RT-mix for each sample was added to 40 μl of the master mix on ice. The PCR consisted of an initial denaturation step at 94°C for 5 min and 30 (BRCA2) or 32 cycles (TBP) of 30 s at 94°C, 30 s at 60°C, 45 s at 72°C, and a final extension step of 10 min at 72°C, using a Perkin-Elmer 9600 DNA thermocycler.
PCR products were diluted tenfold and 2.5-μl aliquots of each were added to 2.5 μl of deionized formamide containing 0.3 μl of a molecular size marker (Genescan 500 ROX, Perkin Elmer). The mixtures were denatured by heating and 2.5 μl aliquots of each were loaded onto 6% polyacrylamide/8 M urea gel and run in the sequencer for 6 h at 1200 V. The different fragments were quantified with Genescan 672 Fragment Analysis software (Perkin Elmer), which calculates the size and area under the peaks. PCR products of target cDNA and corresponding QDS give two different fluorescence peaks. The area under each peak, expressed in relative fluorescence units (RFU), correlates with the amount of PCR product. Results are expressed as a ratio between target cDNA RFU and its corresponding QDS RFU.
Quantitation was based on the determination of the relative amount of the BRCA2 target message to the TBP endogenous control to normalize the amount and quality of total RNA. Final results, expressed as N-fold differences in BRCA2 gene expression relative to TBP gene expression, termed ‘NBRCA2’, were determined as follows:The reproducibility of the quantitative measurements was evaluated by conducting independent replicate cDNA synthesis and PCR reactions.
DNA was extracted from frozen tumour tissue and blood leukocytes of each patient by using standard methods (Maniatis et al., 1982).
The carcinomas were screened with four polymorphic microsatellite DNA marker loci flanking BRCA2 (D13S289, D13S260, D13S171, D13S267) to identify all patients for LOH BRCA2 status.
The PCR was run in a total volume of 50 μl, with 50 ng of genomic DNA, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 400 nM each primer, 200 μM each dNTP and one unit of AmpliTaq DNA polymerase (Perkin Elmer). Microsatellite markers were assayed by PCR amplification of genomic DNA. The annealing temperature, number of amplification cycles and extension time were adapted to each primer set. One microliter of product was mixed with 3 μl of denaturing loading buffer and denatured by heating; then 1.5-μl aliquots of each sample were loaded onto 6% acrylamide gels containing 7.5 M urea. DNA was transferred to nylon membrane filters. The CA repeat probe was labelled with 32P-dCTP by using terminal deoxynucleotidyl transferase. The membrane filters were hybridized overnight at 42°C with the labelled probe, then washed, and autoradiographed at −80°C for an appropriate time.
Normal DNA samples which were polymorphic at a given locus were considered ‘informative’, whereas homozygotes were considered ‘uninformative’. Only cases of constitutional heterozygosity were used in the evaluation of LOH. The signal intensity of the polymorphic alleles was determined by visual examination (three observers) and confirmed by densitometry. The results of all scanned samples were in direct agreement with the initial visual scoring. LOH was considered to occur when the intensity of the allele in tumor DNA was less than 40% of that in corresponding normal-tissue DNA (peripheral blood lymphocytes). LOH was partial in most cases, the band being fainter than the conserved allele but still visible. Such partial losses are due either to contaminating normal tissue or to tumor heterogeneity.
Disease-free survival (DFS) was determined as the interval between diagnosis and detection of the first relapses (local and/or regional recurrences, and/or metastases). Clinical, histological and biological parameters were compared using the chi-square test. Differences between the two populations were judged significant at confidence levels greater than 95% (P<0.05). Survival distributions were estimated by the Kaplan-Meier method (Kaplan and Meier, 1958), and the significance of differences between survival rates was ascertained using the log-rank test (Peto et al., 1977).
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This work was supported by the Comité Régional des Hauts-de-Seine de la Ligue Nationale Contre le Cancer. R Lidereau is a research director with the Institut National de la Santé et de la Recherche Médicale (INSERM). We thank A Khodja for excellent technical assistance. We also thank the Centre René Huguenin staff for assistance in specimen collection and patient care.
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Bièche, I., Noguès, C. & Lidereau, R. Overexpression of BRCA2 gene in sporadic breast tumours. Oncogene 18, 5232–5238 (1999). https://doi.org/10.1038/sj.onc.1202903
- breast carcinoma
- quantitative RT – PCR
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